Contact-killing antimicrobial devices

ABSTRACT

Contact killing antimicrobial articles, devices and formulations are described which kill microorganisms on contact. The articles, devices or formulations contain a non-leaching antimicrobial material which is a unique combination of an organic matrix having biocidal metallic materials non-leachably associated with the matrix. The antimicrobial material may used to form an antimicrobial coating or layer on a surface of the article or device, or may be dispersed in a vehicle or carrier to form a topical antiseptic or disinfectant, or solid shape having contact killing antimicrobial properties. When a microorganism contacts the article, device, or formulation, the biocidal metallic material is transferred to the microorganism in amounts sufficient to kill it.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/151,495, filedSep. 11, 1998 now U.S. Pat. No. 6,126,931, which is a division of U.S.Ser. No. 08/742,580, filed Oct. 28, 1996, now U.S. Pat. No. 5,817,325,which is a continuation-in-part of U.S. Ser. No. 08/663,269 filed Dec.13, 1996, now U.S. Pat. No. 5,869,073, which is a filing under 35 U.S.C.§ 371 of PCT/US94/14636, filed Dec. 19, 1994, which claims priority toU.S. Ser. No. 08/220,821, filed Mar. 31, 1994, now abandoned, and toU.S. Ser. No. 08/170,510, filed Dec. 20, 1993, now U.S. Pat. No.5,490,938, the disclosures of which are herein incorporated byreference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. 2R44EY10787-02, 5 R44 EY10787-03 and 3 R44 EY10787-03S1, awarded by theDepartment of Health and Human Services (National Institutes ofHealth—National Eye Institute). The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to devices coated with or containingunique non-leaching antimicrobial materials capable of killingmicroorganisms on contact, as well as methods of manufacture and use ofsuch materials.

BACKGROUND OF THE INVENTION

The constant threat of bacterial contamination and the associatedrepercussions on health have made preservatives a ubiquitous part ofdrugs and packaged food. However, preservatives oftentimes haveundesirable side effects, especially in pharmaceutical products. Growingconsumer awareness about the deleterious effect of preservatives inrecent years has necessitated their reduction or preferably, totalelimination, without risking bacterial contamination, thus prompting theneed for the development of new, cost effective packaging and storingmethods that prevent bacterial contamination. The problem is acute inthe pharmaceutical area, especially in the ophthalmic industry, which ispresently driven by the need to address the issue of patient sensitivitytoward preservatives in ocular solutions. Burnstein, N. L. et al.,Trans. Ophthalmol. Soc. 104: H02 (1985); Collins, H. B. et al., Am. J.Optom. & Physiolog. Optics, 51: 215 (A89). Similar problem, exist in thefood, medical device, healthcare and water purification areas.

The modality of action of all infection resistant surfaces presentlyknown is via one of the following mechanisms: (i) dissolution of anantimicrobial component into the contacting solution, or (ii) chemicallybound antimicrobials. The former is accomplished by blending anantimicrobial compound with a plastic material. The composite materialis then either molded into a device or applied as a coating. Thebactericidal action of such coatings depend on diffusion of the biotoxicagent into solution. Numerous examples of this type have been reportedin the literature. Another variant of this type involves hydrolysis ordissolution of the matrix containing an antimicrobial compound, therebyeffecting it's release into solution. High levels of preservatives are,however, released into contacting solutions in long term applications.In the latter mechanism, a bioactive compound is covalently bound eitherdirectly to the substrate surface or a polymeric material that forms anondissolving surface coating. The antimicrobial compounds in suchcoatings exhibit greatly diminished activity, unless assisted byhydrolytic breakdown of either the bound antimicrobial or the coatingitself. In either case, relatively high levels of preservative has to bereleased into solution in order to elicit antimicrobial action.

Various products for use externally or internally with humans or animalscan serve to introduce bacterial, viral, fungal or other undesirableinfections. Such products include medical devices, surgical gloves andimplements, catheters, implants and other medical implements. To preventsuch contamination, such devices can be treated with an antimicrobialagent. Known methods of preparing an infection-resistant medical deviceshave been proposed in U.S. Pat. Nos. 3,566,874; 3,674,901; 3,695,921;3,705,938; 3,987,797; 4,024,871; 4,318,947; 4,381,380; 4,539,234;4,612,337; 3,699,956; 4,054,139; 4,592,920; 4,603,152; 4,667,143 and5,019,096. However, such methods are complicated and unsatisfactory.Prior known antimicrobial coatings often leach material into thesurrounding environment. Many are specifically designed for releasingantimicrobial agents (see, U.S. Pat. No. 5,019,096). There is a need formedical devices and other products which are able to resist microbialinfection when used in the area of the body to which they are applied,which provide this resistance over the period of time, and which do notleach antimicrobial materials into the environment.

SUMMARY OF THE INVENTION

It is an object of the invention to provide devices, articles andsurfaces coated with and/or containing contact killing non-leachingantimicrobial materials which are capable of killing microorganisms oncontact, but which do not leach significant amounts of antimicrobialmaterials into the surrounding environment. The antimicrobial materialsmay be deposited on the surface of a substrate to form a contact-killingantimicrobial coating on the surface, may be cast into a freestandingantimicrobial film, or may be incorporated into a carrier to provide abulk antimicrobial which can be applied as desired to form acontact-killing antimicrobial layer.

The articles, devices and surfaces of the present invention are coatedwith, or contain (for example, dispersed throughout the article)antimicrobial materials which are molecularly designed to enable amatrix bound biocide to retain high activity without elution of anycompounds into contacting solutions, carriers or other materials. Theantimicrobial's activity stems from the sustained, cooperative biocidalaction of its components. Selective transfer of one component fromwithin the matrix directly to the microorganism upon contact is achievedvia a “hand off” mechanism upon engagement and penetration of themicroorganism's cell membrane. The antimicrobial material, therefore,maintains long term efficacy without releasing toxic elutables into thesurrounding environment.

The antimicrobial material of the present invention comprises acombination of an organic material which forms a matrix, and a broadspectrum biocide intercalated in the matrix that interacts sufficientlystrongly with the organic material that the biocide does not leach fromthe matrix. The organic material must possess two important properties:it must be capable of reversibly binding or complexing with the biocide,and must be capable of insinuating the biocide into the cell membrane ofthe microorganism. The organic material preferably is capable ofdissolving into or adhering to the cell membrane surrounding themicroorganism. Preferred organic materials are those which can beimmobilized on a surface and which bind the biocide in such a manner asto permit release of the biocide into the microorganism but not into thesurrounding environment. The biocide preferably is a low molecularweight metallic material that is toxic to microorganisms and is capableof complexing with or reversibly binding to the organic matrix material,but which binds preferentially to cellular proteins of microorganisms.When a microorganism contacts the antimicrobial material, the organicmaterial engages or penetrates at least the outer portion of the lipidbilayer of the microorganism's cell membrane sufficiently to permitinsinuation of the biocide into the microorganism, where cell proteinsor proteins in the lipid bilayer compete effectively for the biocide dueto favorable binding constants. The result is a contact-killing deliverysystem that selectively transfers the biocide through or into themicroorganism's cell membrane upon contact without elution of thebiocide into solution, thereby maintaining long term efficacy. Theunique mode of action of the presently described antimicrobial materialoffers high surface activity coupled with substantially low leachables.

The invention provides for the first time cooperative bioactivity in acontact killing, nonleaching system.

Organic materials useful in the present invention comprise materialswhich are capable of: 1.) reversibly binding or complexing with abiocide, and 2.) insinuating the biocide into the cell membrane of themicroorganism upon contact. A preferred class of materials are thosehaving the aforementioned properties, and which are capable ofcomplexing and/or binding a bactericidal metallic material. Mostpreferred is the class of organic materials which can dissolve into, oradhere to, and penetrate at least the outer portion of the lipid bilayermembrane of a microorganism. For this purpose, surface active agents,such as cationic compounds, polycationic compounds, anionic compounds,polyanionic compounds, non-ionic compounds, polynonionic compounds orzwitterionic compounds are useful. Organic materials which currently aremost preferred for use in the invention include cationic or polycationicmaterials such as biguanide compounds.

In a preferred embodiment of the present invention, the organic materialis crosslinked to form the matrix. Cross-linking agents which can beused in the present invention are those which react with thepolycationic material to form a crosslinked network or matrix. Suitablecrosslinking agents include, for example, organic multifunctional groupssuch as isocyanates, epoxides, carboxylic acids, acid chlorides, acidanhydrides, succimidyl ether aldehydes, ketones, alkyl methane sulfones,alkyl trifluoromethane sulfonates, alkyl paratoluene methane sulfones,alkyl halides and organic multifunctional epoxides. In a currentlypreferred embodiment, the organic material comprises a polyhexamethylenebiguanide polymer which is crosslinked with an epoxide, such asN,N-bismethylene diglycidylaniline, to form a crosslinked network ormatrix.

The biocidal material can be any antimicrobial material which is capableof non-leachably binding to or complexing with an organic matrix, butwhich, when placed in contact with the microorganism, preferentiallytransfers to proteins in the microorganism. For this purpose, metallicmaterials which bind to cellular proteins of microorganisms and aretoxic to the microorganisms are preferred. The metallic material can bea metal, metal oxide, metal salt, metal complex, metal alloy or mixturethereof. Metallic materials which are bactericidal or bacteriostatic arepreferred. By a metallic material that is bacteriostatic or bactericidalis meant a metallic material that is bacteriostatic to a microorganism,or that is bactericidal to a microorganism, or that is bactericidal tocertain microorganisms and bacteriostatic to other microorganisms.Examples of such metals include, e.g., silver, zinc, cadmium, lead,mercury, antimony, gold, aluminum, copper, platinum and palladium, theirsalts, oxides, complexes, and alloys, and mixtures thereof. Theappropriate metallic material is chosen based upon the use to which thedevice is to be put. The preferred metallic materials are silvercompounds. In a currently preferred embodiment, a silver halide is used,most preferably, silver iodide.

The invention further comprises liquid compositions for forming acontact killing, non-leaching antimicrobial layer or coating on asurface. In one embodiment, the composition is a two-part compositioncomprising a first solution dispersion or suspension of an organicmaterial, and a second solution, dispersion or suspension of a biocidalmaterial. If a crosslinked coating or film is desired, the firstsolution, dispersion or suspension also will contain the crosslinkingagent. As a first step, the crosslinking agent and the organic materialmay be reacted to form a non-crosslinked adduct. To form acontact-killing nonleaching coating or layer on a substrate, the firstcomposition is applied to the substrate under conditions sufficient toimmobilize the organic material on the substrate, forming a matrix. If acrosslinking agent is present, the matrix is cured to inducecrosslinking. The matrix then is exposed to the solution of the biocidalmaterial under conditions sufficient induce the biocidal material tobecome non-leachably attached to, complexed with or associated with thematrix.

In another embodiment, the liquid composition is a one part compositioncomprising a solution, dispersion or suspension of the organic material,the biocidal material, and optionally, the crosslinker. To form thecontact-killing coating on a substrate, this composition is applied tothe substrate under conditions sufficient to immobilize the organicmaterial on the substrate, forming a matrix in which the biocidalmaterial is non-leachably attached to or associated with the matrix.

Both the two part composition and the one part composition also may beused to make freestanding antimicrobial films, as described in moredetail below. As used herein, the term “freestanding” means not attachedto a substrate.

The invention further provides methods for making the compositions ofthe present invention, and applying them to various substrates to formantimicrobial coatings or layers on the substrates. The method formaking the composition generally comprises providing a solution,dispersion or suspension an organic material, and, if a non-crosslinkedmaterial is desired, applying the solution, dispersion or suspension ofthe organic material to the substrate to form the matrix. The organiccompound may be attached to and/or immobilized on the substrate by anyappropriate method, including covalent bonding, ionic interaction,coulombic interaction, hydrogen bonding, crosslinking (e.g., ascrosslinked (cured) networks) or as interpenetrating networks, forexample. If a crosslinked coating is desired, the organic material firstis combined with a crosslinking agent. Typically, both the organicmaterial and the crosslinker will be in liquid form (e.g., in asolution, dispersion or suspension), and the two solutions are combined,forming a liquid mixture. The liquid may be an organic solvent, anaqueous liquid or a mixture of an organic solvent and an aqueous liquid.The organic material and the crosslinking agent then are reacted to forman adduct. The resulting adduct can be stored for later use, if desired,or can be immediately applied to a substrate. The organic material (withor without the added crosslinker) can be applied to the substrate ofchoice by any suitable means for applying a liquid coating, including,for example, spraying, brushing, dipping, calendering, rod or curtaincoating. The method selected to apply the composition to the substratewill depend on several factors, including the coating thickness desiredand the nature and configuration of the substrate. If necessary, thesurface to be coated is cleaned or treated before the polymer solutionis applied. The resulting coating is dried to form the matrix, or, if acrosslinker is present, subjected to crosslinking conditions, forming acrosslinked network. Crosslinking conditions may include thermal curing,ultraviolet curing, chemical curing or other curing methods. The matrixthen is contacted with a solution of the biocidal material underconditions sufficient to deposit the biocidal material into the matrixsuch that the biocidal material becomes non-leachably associated with orattached to the matrix.

Another embodiment of the method of making the compositions and coatingsof the present invention comprises first combining the organic materialand the biocidal material, then applying the mixture to the substrate toform the matrix as described above. If a crosslinked coating is desired,the organic material and crosslinking agent are combined and reacted toform an adduct as described above, then the adduct is combined with thebiocidal material. The resulting adduct/biocide mixture can be storedfor later use, or can be immediately applied to a substrate and cured asdescribed above to induce crosslinking, thereby forming the polymericnetwork having the biocidal material non-leachably associated therewithor attached thereto.

In the methods of the invention described above, the amounts and/orconcentrations of the materials used will depend upon the nature andstoichiometry of the materials used, and the end product desired. In thecurrently preferred embodiments, the concentration of the solution,dispersion or suspension of the organic material, or the organic adductresin formed by the reaction of the polymer and crosslinker, typicallyis in the range of from about 0.5 to about 20% by weight. Typically, apolymer:crosslinker ratio in the range of from about 1:1 to about 3:1(weight percent) will form crosslinked networks which will non-leachablyretain the biocide and preferentially transfer the biocide to themicroorganism upon contact as described herein. Solutions of thebiocidal material typically comprising from about 0.005 to about 0.5% byweight can be used to impregnate the matrix with biocide.

In another embodiment of the present method, a freestandingantimicrobial film may be formed using the present antimicrobialmaterial. In this embodiment, using the two-part compositions describedabove, a solution, suspension or dispersion of the organic material iscast on a non-adherent substrate and dried to form a film. If acrosslinked material is desired, the organic material and crosslinkerfirst are combined and reacted to form an adduct as described above, anda solution, suspension or dispersion of the adduct is cast to form thefilm. The film is cured to induce crosslinking, as described above. Thefilm then is contacted with a solution, dispersion or suspension of thebiocidal material to deposit the biocidal material within the matrix oforganic material. The film then is detached from the substrate and usedas desired. Alternatively, freestanding crosslinked or non-crosslinkedfilms can be cast using the one part liquid compositions describedabove. In this embodiment, the organic material must have film formingcapability so that a coherent film can be obtained free of anysubstrate. Freestanding antimicrobial materials also may be preparedusing the antimicrobial materials of the present invention in otherphysical forms besides films, such as microbeads or solid shapes, forexample.

In another embodiment, an antimicrobial powder may be formed by castinga freestanding film, as described above, then grinding the film to apowder. The powder has the same contact-killing antimicrobial propertiesas the films and coatings described above. The antimicrobial powder canbe incorporated into a carrier, such as a gel, cream or liquid, andapplied to a surface to form an antimicrobial layer. For example, aformulation comprising the antimicrobial powder dispersed in apharmaceutically acceptable carrier can be used as a topical antisepticand be applied to a wound. The powder also can be dissolved or dispersedin a carrier or vehicle which can be spread, sprayed, wiped or appliedin some other manner to form a contact killing antimicrobial layer on asurface or to kill microbes on the surface. The powder also may becompounded into a polymeric material and cast or molded into a solid orsemisolid article. The resulting solid or semisolid article has contactkilling antimicrobial capability.

In a preferred embodiment, the antimicrobial materials of the presentinvention is used to form a contact-killing surface on a substrate. Toprovide the contact-killing surface on the substrate, the organiccompound may be attached to and/or immobilized on the substrate by anyappropriate method, including covalent bonding, ionic interaction,coulombic interaction, hydrogen bonding, crosslinking (e.g., ascrosslinked (cured) networks) or as interpenetrating networks, forexample. In a currently preferred embodiment, the organic matrix isformed by first reacting polyhexamethylenebiguanide withN,N-bismethylene diglycidylaniline to form an adduct. Stable coatingsolutions of the resulting adduct have been obtained in both absoluteethanol and in aqueous ethanol. The adduct can be applied on a substratesurface either by dip-coating, brushing or spraying. Once applied to thesubstrate, the coating is thermally cured to induce crosslinking,thereby forming a polymeric network on the substrate. The resultingcoating is optically clear, resistant to most solvents and totemperature changes, and does not delaminate, flake or crack. Thecoating typically is about ten microns or less in thickness, althoughthe thickness of the coating may be varied by well-known techniques,such as increasing the solids content of the resin. A broad spectrummetallic antimicrobial, preferably a silver compound, then is introducedinto the polymeric network such that it is entrapped as submicronparticles, and complexes with the functional groups on the polymer. Inthe currently preferred embodiment, the broad spectrum antimicrobial isa silver halide, preferably silver iodide.

The antimicrobial materials of the present invention are unique in thefollowing respects:

-   i) The unique nature of the antimicrobial coating material utilizes    a cooperative effect of it's components. This results in high    biocidal activity, while maintaining almost no significant    leachables into solutions it is in contact with.-   ii) The mechanism of action is essentially a surface mediated one,    whereby organisms succumb only upon contact with the material due to    the non-leaching property associated with it.-   iii) The ability of such surfaces to remain completely inert in    solution in the absence of microorganism contamination.-   iv) The ability of such surfaces to remain viable over multiple    organism challenges with no decrease in their bioactivity.-   v) The utilization of such biocidal materials on an interior or    exterior surface of a device, thereby eliminating the possibility of    microbial colonization on the surface.-   vi) User friendliness and cost effectiveness of the coating for all    types of applications.-   vii) Adaptability to existing manufacturing technology, thereby    enabling large scale manufacture with minimal cost.-   viii) Applicability to a variety of liquid formulations over a wide    range of solution viscosity including artificial tears, saline,    anti-glaucoma and ocular hypertensension drugs, and contact lens    cleaning solutions.-   ix) Readily adaptable for the delivery of other types of medicaments    or solutions where preservatives have been used such as ear and    nasal drug formulations.

The above and other objects, features and advantages of the presentinvention will be better understood from the following specificationwhen read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic graphic illustration of the matrix/biocidecomplex of the present invention, before contact of the coating withmicroorganisms.

FIG. 1B is a schematic graphic illustration of the contact-killingability of the matrix/biocide complex of the present invention duringcontact of the coating with microorganisms.

FIGS. 2A-D are a schematic graphic illustration of a preferred methodfor applying the matrix/biocide complex of the present invention to asubstrate:

FIG. 2A shows the matrix immobilized on the substrate, with chains ofthe organic material forming arms or tentacles that protrude into thesurrounding environment;

FIG. 2B shows the immobilized matrix impregnated with a biocidalcompound, with reservoirs of the biocide deposited within the matrix andmolecules of the biocidal compound attached to the tentacles;

FIG. 2C shows a microorganism in contact with the matrix/biocide complexwherein the polymer chains engage and dissolve into the microorganismcell membrane;

FIG. 2D shows penetration of the cell membrane and transfer of thebiocide from the network into the microorganism, causing cell death.

FIG. 3 is a graph illustrating the bioactivity of a preferred coating ofthe present invention, a matrix formed from crosslinked PHMB complexedwith silver salts, treated as a function of surface area to volumeagainst Pseudomonas aeruginosa microorganisms in phosphate bufferedsaline at 30° C.

DETAILED DESCRIPTION

The contact-killing non-leachable antimicrobial materials of the presentinvention can be applied to a variety of substrates. Antimicrobialcoatings according to the present invention can be applied, for example,to woods, metals, paper, synthetic polymers (plastics), natural andsynthetic fibers, natural and synthetic rubbers, cloth, glasses, andceramics. Examples of synthetic polymers include elastically deformablepolymers which may be thermosetting or thermoplastic such as, forexample, polypropylene, polyethylene, polyvinylchloride, polyethyleneterephthalate, polyurethane, polyesters, rubbers such as polyisoprene orpolybutadiene, polytetrafluoroethylene, polysulfone and polyethersulfonepolymers or copolymers. The substrate can be a deformable metallic orplastic medicament container, such as a toothpaste tube, where thecontainer may remain deformed after each dose is dispensed. Otherpolymeric materials, including polymeric materials which are used forthe preparation of membranes or filter papers, also can serve assubstrates. Examples of organic polymeric materials include polyamide(e.g., nylon), polycarbonate, polyacrylate, polyvinylidene fluoride,cellulosics (e.g., cellulose), and Teflon®. The substrate can be eitherhydrophilic or hydrophobic. With the exception of silicone and Teflon®,which may require prior surface activation with techniques such asplasma, chemical oxidation or metallic sensitization, e.g., a primer, nosurface activation is necessary. Inorganic materials to which thepresent coatings can be applied include glass fiber materials, ceramicssuch as alumina or silica, and metals. Sintered glass and sinteredceramic substrates also can be used.

The term “microorganism” as used herein includes bacteria, blue-greenalgae, fungi, yeast, mycoplasmids, protozoa and algae.

The term “biocidal” as used herein means bactericidal or bacteriostatic.The term “bactericidal” as used herein means the killing ofmicroorganisms. The term “bacteriostatic” as used herein meansinhibiting the growth of microorganisms, which can be reversible undercertain conditions.

As used herein, the terms “non-leachable” or “substantiallynon-leachable” means that none or very minute amounts (e.g., below acertain threshold) of the organic and/or biocidal material dissolvesinto a liquid environment. Preferably, this threshold is no higher than1 part per million (ppm), and more preferably is lower than 100 partsper billion (ppb).

Organic materials useful in the present invention comprise materialswhich are capable of: 1.) reversibly binding or complexing with thebactericide, and 2.) insinuating the bactericide into the cell membraneof the microorganism. A preferred class of materials are those havingthe aforementioned properties, which are capable of being immobilized ona surface and which preferentially bind a bactericidal metallic materialin such a manner so as to permit release of the metallic biocide to themicroorganism but not to the contacting environment. Most preferred isthe class of organic materials which can dissolve into, adhere to,disrupt or penetrate the lipid bilayer membrane of a microorganism. Forthis purpose, surface active agents, such as cationic compounds,polycationic compounds, anionic compounds, polyanionic compounds,non-ionic compounds, polyanionic compounds or zwitterionic compounds maybe used. Organic materials which currently are most preferred for use inthe invention include cationic or polycationic compounds such asbiguanide compounds. These may be attached to and immobilized on asubstrate, or used to form the matrix of a freestanding film, by anyappropriate method, including covalent bonding, ionic interaction,coulombic interaction, hydrogen bonding, crosslinking (e.g., ascrosslinked (cured) networks) or as interpenetrating networks, forexample.

Preferred cationic materials include benzalkoniumchloride derivatives,a-4-[1-tris(2-hydroxyethyl) ammonium-2-butenyl]poly[1-dimethylammonium-2-butenyl]-ω-tris(2-hydroxyethyl) ammoniumchloride, and biguanides of the general formula:

or their water soluble salts, where X is any aliphatic, cycloaliphatic,aromatic, substituted aliphatic, substituted aromatic, heteroaliphatic,heterocyclic, or heteroaromatic compound, or a mixture of any of these,and Y₁ and Y₂ are any aliphatic, cycloaliphatic, aromatic, substitutedaliphatic, substituted aromatic, heteroaliphatic, heterocyclic, orhetero aromatic compound, or a mixture of any of these, and where n isan integer equal to or greater than 1. Preferred compounds include,e.g., chlorhexidine (available from Aldrich Chemical Co., Milwaukee,Wis.) or polyhexamethylene biguanide (available from Zeneca Biocides,Inc. of Wilmington, Del.). The above-mentioned organic materials may bemodified to include a thiol group in their structure so as to allow forthe bonding of the compound to a metallic substrate, or may bederivatized with other functional groups to permit direct immobilizationon a non-metallic substrate. For example, the above-mentioned organicmaterials may be suitably functionalized to incorporate groups such ashydroxy, amine, halogen, epoxy, alkyl or alkoxy silyl functionalities toenable direct immobilization to a surface.

In a preferred embodiment of the present invention, the organic materialcomprises a polycationic material which is crosslinked to form thematrix. Crosslinking agents which can be used in the present inventionare those which react with the polycationic material to form acrosslinked network or matrix. Suitable crosslinking agents include, forexample, organic multifunctional groups such as isocyanates, epoxides,carboxylic acids, acid chlorides, acid anhydrides, succimidyl etheraldehydes, ketones, alkyl methane sulfones, alkyl trifluoromethanesulfonates, alkyl paratoluene methane sulfones, alkyl halides andorganic multifunctional epoxides. In a currently preferred embodiment, apolyhexamethylene biguanide polymer is crosslinked with an epoxide, suchas N,N-bismethylene diglycidylaniline, to form a crosslinked network.

The biocidal material can be any antimicrobial material which is capableof non-leachably binding to or complexing with the organic matrix, butwhich, when placed in contact with the microorganism, preferentiallytransfers to the microorganism. For this purpose, metallic materialswhich are toxic to microorganisms are preferred. The metallic materialcan be a metal, metal oxide, metal salt, metal complex, metal alloy ormixture thereof. Metallic materials which are bactericidal orbacteriostatic are preferred. By a metallic material that isbacteriostatic or bactericidal is meant a metallic material that isbacteriostatic to a microorganism, or that is bactericidal to amicroorganism, or that is bactericidal to certain microorganisms andbacteriostatic to other microorganisms. Examples of such metals include,e.g., silver, zinc, cadmium, lead, mercury, antimony, gold, aluminum,copper, platinum and palladium, their oxides, salts, complexes andalloys, and mixtures of these. The appropriate metallic material ischosen based upon the use to which the device is to be put. Thecurrently preferred metallic materials are silver compounds.

The biocidal material can be introduced into the matrix eithercontemporaneously with or after application of the organic material to asurface.

The invention also provides for a substrate in which the surface is atleast partially coated with additional organic materials, and/orbiocidal materials, or both. Examples of organic and biocidal materialsthat can be used are discussed above. The use of a combination of atleast two different organic and biocidal materials can enhance theantimicrobial properties of the coating. Different types ofmicroorganisms can exhibit different degrees of sensitivity to differentorganic and biocidal materials. In addition, the use of two or moredifferent organic and biocidal materials can significantly reduce theproblem of selection for microorganisms having resistance to the organicand biocidal materials in the coating that can occur when only one isused.

The amount and/or type of the antimicrobial coating which is used in aparticular application will vary depending on several factors, includingthe type and amount of contamination which is likely to occur, and thesize of the antimicrobial surface. The amount of antimicrobial used willbe a minimum amount necessary to maintain the sterility of the liquid.As stated above, this amount will vary depending upon variousconsiderations.

In a preferred embodiment, the organic material, whether crosslinked ornon-crosslinked, forms an insoluble, non-leachable matrix having aunique configuration: some of the organic material protrudes into thesurrounding environment, that is, “arms” or “tentacles” of the organicmaterial project away from the matrix and into the surroundingenvironment. This phenomenon can be understood by referring to FIGS. 1and 2, which are schematic graphic illustrations of a preferred coatingof the present invention in which the organic material is a crosslinkedbiguanide polymer and the biocidal material is a silver halide salt,preferably silver iodide. FIGS. 1A and 1B and FIGS. 2A-D show thepolymer matrix having tentacles projecting into the ambient environment,and the silver salt deposited in reservoirs and on the tentacles.Without wishing to be bound by theory, it is believed that when amicroorganism contacts the coating, the biguanide polymer tentaclesdissolve into the lipid bilayer surrounding the microorganism, therebyintroducing silver molecules into the interior of the microorganism orto proteins within the cell membrane. The silver salt has a greateraffinity for certain proteins in the microorganism than for the polymer,and therefore complexes with the cellular proteins and is transferredinto the microorganism, thereby killing it. Specifically, it is thoughtthat the silver forms complexes with the sulfhydryl and amino groups ofthe cellular proteins.

In this embodiment, the silver salt is attached to or impregnated intothe matrix and on the tentacles of the polymer such that the silver issubstantially non-leachable. Again, not wishing to be bound by theory,it is believed that the silver salt forms complexes with functionalgroups in the polymer, and that the complexed silver resists leachinginto ambient liquids or other materials (e.g., creams or gels) incontact with the coated surface. However, when the coating becomesexposed to cellular proteins, the silver preferentially complexes withthe proteins.

In a currently preferred embodiment, the polymeric material ispolyhexamethylene biguanide, (PHMB), the crosslinking agent isN,N-bismethylenediglycidylaniline (BMDGA), and the silver salt is asilver halide, most preferably, silver iodide. In this embodiment, thecoating is made by combining a solution of polyhexamethylene biguanidewith a solution of the crosslinking agent, and reacting the mixtureunder conditions sufficient to form a non-crosslinked PHMB-BMDGA adduct.The ratio of PHMB to BMDGA preferably is in the range of from about 1:1to 3:1 by weight. The PHMB-BMDGA mixture is heated to about 95° C. forabout 2 hours in a closed reactor to form the adduct. The concentrationof the resulting adduct resin preferably is in the range of from about0.5 to about 20% by weight. The adduct resin solution is coated onto thedesired substrate, and heated to a temperature sufficient to inducecrosslinking between the adducts, thereby forming a crosslinked networkor matrix. Temperatures sufficient for crosslinking typically are in therange of from about 70° C. to about 200° C. The resulting crosslinkednetwork is then saturated with silver by immersing the coating for abouttwo minutes in a silver iodide/potassium iodide solution. Silversolutions having a concentration of from about 0.005 to about 0.5% canbe used for this step. The silver iodide forms reservoirs in the matrix,and becomes attached to the tentacles. Silver iodide has sufficientaffinity for the PHMB polymer that it forms an insoluble complex thatwill not leach into ambient solutions or other materials in contact withthe material, even at elevated temperatures. However, when amicroorganism contacts the coating, the tentacles disrupt themicroorganism's lipid bilayer membrane, thereby introducing the silveriodide into the microorganism. Silver iodide has greater affinity forcertain proteins within the microorganism than for the PHMB-BMDGAmatrix, and forms complexes with these proteins, that is, the silver ispreferentially transferred from the coating to the microorganism. Thesilver accumulates to toxic levels in the microorganism and kills it.The silver iodide reservoirs within the matrix replenish the silveriodide on the tentacles lost to the microorganism by reestablishing theequilibrium for formation of the complex (AgI+PHMB⇄[PHMBAgI]).

This invention also includes the coated substrates, freestanding films,microbeads or other shapes, powders and articles made in accordance withthe above methods.

The present invention provides stable, adherent coatings or layers usingthe present coating formulations on a wide range of materials, includingthose commonly used in membranes and in medical device manufacture. Thecoating or layer may be applied directly to most surfaces without priorsurface modification. Studies simulating a year of contact between thecoating and aqueous solutions at ambient temperature resulted in lessthan 100 ppb of any active ingredient in the solution. The extractsolutions themselves (solutions which have been in contact with thecoating) show no antimicrobial or mammalian cell toxicity. The coatedsurface remains fully inert and bio-active after exposure to variousphysical and chemical stresses including: low temperature, ethanol,boiling water, prolonged exposure to varying pH solutions and solutionsof high ionic strength, as well as sterilization by conventional methods(e.g., wet autoclave, ethylene oxide, γ-irradiation, ethanol).

Surface coatings, freestanding films, beads and articles andformulations containing the antimicrobial powder or microbeads accordingto the present invention exhibit antimicrobial activity against bothgram positive and gram negative bacteria and yeast, and are resistant tofungal growth. Treated surfaces completely kill organisms at challengelevels of 10⁶-10⁸ CFU/mL within 8 to 20 hours at 30° C., depending onorganism type. Tables 1 and 2 (in Example 4) list the bioactivity ofcoated surfaces towards different challenge organisms. The coatingrenders surfaces biofilm resistant, which coupled with its chemicalinertness, makes it particularly suited for many device applications.

The antimicrobial materials of the present invention also have beensuccessfully applied on the surface of microporous membranes, includingwithin the pores as evidenced by SEM-EDX. Stable, uniform coatings havebeen obtained on a variety of membrane materials with almost noreduction in their flow property. Coated microporous membranes killmicro organisms upon contact and are resistant to the phenomenon of“bacterial grow-through” which occurs even in sterilizing 0.2 μM poresize membranes in long term contact applications. Such membranes are,therefore, well suited for incorporation in devices used in long termfiltration applications such as multi-dose dispensers for preservativefree formulations, water purification systems and in any applicationwhere it is desirable to use barrier properties of a membrane for morethan a day.

The mechanism of action is one wherein the antimicrobial materials areactivated only upon contact with the microorganism. Once themicroorganism accumulates a toxic amount of silver, it succumbs anddetaches from the surface. The coating or other treated surface,therefore, remains active only as long as viable organisms contact it,and reverts to being inert in their absence. This unique propertywhereby the biological activity is triggered by bacterial cell contactenables the coating to function “intelligently.” For such a contactmechanism to be valid, the rate of kill is expected to vary as afunction of the ratio of total surface area of coated substrate to thevolume of the bacterial suspension in contact with it (S/V ratio) atconstant temperature. As shown in the Examples, time to kill experimentswere performed on coated polyethylene tubes of varying inner diameterthat were inoculated with predetermined volumes of a suspensioncontaining 10⁶ CFU/mL of Pseudomonas aeruginosa in phosphate bufferedsaline (PBS). The decrease in organism concentration was measured as afunction of time at constant temperature over 20 hours. Experimentalresults are summarized in FIG. 3. There is no substantial difference inkill rate for S/V ratios ranging from 2.5 to 5 cm⁻¹; similar resultswere obtained for a ratio of 1.5. For the largest diameter tubes tested(S/V 0.5), however, viable organisms were detected at low levels, whichcan be attributed to a decrease in probability of organisms contactingthe surface with increasing volumes. No toxic components were found inorganism-free solutions in contact with coated tubes under identicalconditions when tested both chemically and biologically, which supportsthe proposed contact mechanism for cell death. Such a distinction wouldnot be evident if sterilization were to occur via either controlleddissolution or diffusive elution of the coating components intosolution; in either case, high levels of active components would bepresent in solution.

The antimicrobial materials of the present invention can be used to formcontact-killing coatings or layers on a variety of substrates. As shownin the Examples, the material forms a non-leaching contact-killingsurface on medical devices such as catheters, urological devices, bloodcollection and transfer devices, tracheotomy devices, intraocularlenses, and on personal care products such as toothbrushes, contact lenscases and dental equipment. The materials can be used on medical devicesand healthcare products, baby care products, personal hygiene products,household products, food preparation surfaces and packaging, waterstorage, treatment and delivery systems, biosensitive systems andlaboratory and scientific equipment.

In addition to forming coatings or layers on substrates, the presentantimicrobial materials can be used to makes antimicrobial powders,microbeads, or solid shapes. The present antimicrobial materials inpowder form or in the form of microbeads, for example, can be dispersedor dissolved in a carrier and used as a topical antiseptic, wounddressing or topical disinfectant. The present antimicrobial materials(e.g., in powder or microbead form) also can be dispersed in a polymericmatrix and used to make plastic articles having contact-killingantimicrobial properties, for example, countertops, shelving material,toilet seats and other bath fixtures, cutting boards and other kitchenmaterials, and many others.

Contact lens cases are a proven contributor to the spread of ocularpathogens and disease. A lens case coated with a coating of the presentinvention has been shown in vitro to kill all clinically relevantpathological strains of micro-organism without leaching toxic chemicalsinto the contact lens solution (see Examples 2 and 4) Once a bio-filmhas formed on an untreated contact lens case, it is resists virtuallyall types of disinfection products currently available for contact lenscare. Thus, the bio-film serves as a reservoir for bacteria thatre-contaminate the lens each time it is stored in the case. The treatedlens case is compatible with all disinfecting solutions tested to date.Use of the coating permits sterilization of the lenses using ordinarysaline as the soaking solution.

The present antimicrobial materials can be used to coat ordinary nylonbristle toothbrushes (see Example 2). The treated toothbrush kills thepathogens commonly found in the human mouth and on bathroom surfaces,while untreated toothbrushes foster their growth. It is believed thattoothbrushes are partly responsible for the spread of oral and dentaldisease. In vitro and in vivo test programs examined the types, numberand kill-rates for the organisms commonly found in the mouth. The testsindicated that the treated toothbrush eliminated virtually all of thesepathogens over a 12 hour period. The inert coating does not elute fromthe brush and therefore has no taste and poses no risk to the consumer.The present materials also can be used to provide an antimicrobial layeror to kill microbes on dental instruments, dental floss, and otherdevices for use in the mouth.

Bio-film formation is a major problem in many water container, waterfiltration, and water delivery applications. Once a bio-film is formed,it typically resists further treatment and acts as a constant source ofmicrobial contamination. Prevention of bio-film formation is key to themaintenance of high quality water systems. The present materials can beused to prevent bio-film formation on many water treatment products. Forexample, water containers and water purification systems used incamping, residential, commercial and military applications, which needto be periodically emptied, disinfected and rinsed. Treatment with thepresent antimicrobial materials would eliminate the costs and hazardsassociated with this process, as well as the risks associated withimproper maintenance of these water storage systems.

The present antimicrobial materials also are useful in point-of-usewater purification filters, which trap bacteria and nutrients commonlyfound in all water systems. The bio-films formed in these filters oftenshed bacteria into the water stream in quantities exceeding the standardsafety limits. Treated filters would offer longer service life andsignificantly reduce the potential for bio-hazard.

Surfaces in medical offices, such as treatment tables, or consoles in atypical dental office have proven to be a major source of bacterialcontamination—posing potential health risks to the patient and staff.Although water supplies are routinely treated to reducebio-contamination, water standing in the lines in the dental console canpromote the formation of bio-films. Coating or treating these surfaceswith the antimicrobial materials of the present invention can preventbio-film formation on these substrates.

The present antimicrobial materials have been tested against thebacteria most commonly found in water. Treated tubing withstood repeatedattempts to bio-film formation at very high challenge levels, whileuntreated control tubing developed extensive bio-film (see Example 5).The treated tubing showed no traces of chemical elution into the water.

The present materials also can be applied to woven and non-woven fabricsused in hospitals and on healthcare supplies ranging from face masks tobed sheets. The materials can be applied in a spray or wipe form whichcan be applied to surfaces in order to make them antimicrobial.

Long term indwelling catheters pose a risk of infections (2%-9%) whichincreases patient discomfort, the risk of systemic infections and thelength of the patient's hospital stay. Catheters treated with thepresent antimicrobial materials can reduce the presence of infectioncausing bacteria. The materials also can be used on urinary catheters,implants and inserts designed to deal with incontinence suffer fromincreased risk of infection. Coatings made with the present materialshave been demonstrated to kill microorganism in human urine.

The antimicrobial materials of the present invention can be used totreat standard biological plastic laboratoryware for applications whichrequire low microbiological contamination, e.g. cell culture lab ware.

EXAMPLES Example 1 Preparation of PHMB-BMDGA Solutions

Polyhexamethylene biguanide (PHMB) (available as a 20% aqueous solutionfrom Zeneca Biocides, Wilmington, Del.) was distilled to remove thewater, and the PHMB was re-dissolved in absolute ethanol to give a 20%by weight solution. This solution was used to prepare the resinsoutlined below.

(a) 312 mL of the 20% PHMB solution in ethanol was further diluted with600 ml of ethanol. This solution was added to a solution ofN,N-bismethylene diglycidylaniline) (BMDGA) (Aldrich Chemical Company,Milwaukee, Wis.) containing 37.60 grams of BMDGA dissolved in 119.9 mlof acetonitrile and 280.1 ml of ethanol. The resulting mixture washeated at 95° C. in a closed reactor for two hours, forming a PHMB-BMDGAadduct. The adduct solution was cooled and filtered (Scientific Grade417 filter). The resulting adduct solution contained 10% by weight ofPHMB:BMDGA adduct having a PHMB:BMDGA ratio of 1.5:1.(b) 330 μL of the 20% PHMB. This solution was combined with 100 ml of asodium hydroxide (NaOH) solution containing 66 grams of NaOH, 66 ml ofwater and 34 ml of ethanol. This mixture was added to a solutioncontaining 40 grams of acetonitrile and 280 ml of ethanol. The resultingsolution was heated at 95° C. for 2 hours forming the PHMB-BMDGA adduct.The solution was cooled and filtered as described above. The resultingadduct solution contained 10% by weight of PHMB:BMDGA adduct having aPHMB:BMDGA ratio of 1.5:1.

The resins were characterized according to the following procedures:

-   1. Film formation was tested by a dip test with PE/PP    (polyethylene/polypropylene) in which PE/PP samples were dipped in    the resin solutions made in (a) and (b) above and dried by hot air a    blowing, and film formation was observed;-   2. The ratio of polymer to crosslinker (PHMB-BMDGA) in the resin    solution was tested by UV/visible spectroscopy;-   3. Gelation time of the resin mixture was tested.

The resins were diluted with ethanol to a concentration of 1%. Filmformation of the diluted resins were tested by the dip test with PE/PPas described above. Both resins formed a coherent film. The resins werestored in closed containers at ambient temperature.

Example 2 Coating of Plastic Articles

Various plastic articles were coated using the coating solutionsdescribed in Example 1.

-   1. contact lens cases: polyethylene and polypropylene contact lens    cases were coated according to the following procedure:

The contact lens cases were cleaned by immersing them in absoluteethanol for 5 minutes and dried. The cleaned cases were immersed in theantimicrobial coating solution (Example 1a or 1b) for 1 to 2 minutes.The sample cases were dried by hot air blowing. Crosslinking was inducedby heating the cases at 120° C. for the polyethylene cases and at 200°C. for the polypropylene cases for 2 hours. The cases were allowed tocool, rinsed with 60° C. water to remove any unbound polymer, then driedat 60° C. for 1-3 hours.

The coated cases were immersed in a 0.05% solution of silveriodide/potassium iodide in alcohol for 2 minutes. The cases were rinsedwith aqueous alcohol to remove any unbound silver. The cases then wererinsed with water and dried.

-   2. Toothbrush bristles: toothbrushes with nylon bristles were coated    according to the procedure described for contact lens cases, except    that the cross-linking reaction was carried out at 120-140° C.-   3. Polyurethane and Polyvinylchloride Catheters: polyurethane and    polyvinyl chloride catheters were coated according to the procedure    described for contact lens cases, except that the crosslinking    reaction was carried out at 80-120° C. for polyurethane and at    120° C. for polyvinylchloride.-   4. Dental Water Line Unit Tubing And Filters: polyurethane tubing    and polyethersulfone membrane and housing were coated according to    the procedure described for contact lens cases, except that the    crosslinking reaction was carried out at 80-120° C. for polyurethane    and 120-140° C. for polyethersulfone.-   5. Coating Process for Silicone Parts: The parts were pre-cleaned in    100% ethyl alcohol (reagent grade) to remove dirt, grease and other    contaminants. They are then subjected to an alkaline etch by    immersing them in a 0.1M NaOH in 90% ethanol solution (10% water) at    room temperature and ultrasonicated for 2 minutes. They were then    coated in an identical manner as the contact lens cases.-   6. Coating Process for Teflon Parts: The parts were subjected to    surface pretreatment by oxygen plasma for 5 minutes in a plasma    reactor. They were then coated in an identical manner as the contact    lens cases.-   7. Coating Process for Nylon Sheets: Nylon sheets were pre-cleaned    with 100% ethyl alcohol (reagent grade) to remove dirt, grease and    other contaminants. The one part formulation of coating resin has    been diluted with 100% ethyl alcohol to the desired concentration of    1 wt. %. The cleaned nylon sheet was immersed in the coating resin    for a period of 1-2 mins. Then, the sheet was carefully removed from    the coating resin bath and the excess adhering resin was allowed to    drain off. The coating on the nylon sheets was dried by placing them    in an oven at 70° C. for 3-4 mins. Then, dried resin coating was    then crosslinked by thermal curing at 120° C. for a period of 2    hours. The cured samples were removed from the oven and allowed to    cool to room temperature. This procedure was used to coat nylon    toothbrush bristles, non-woven nylon and cellulose fibers.

Example 3 Membrane Coating Procedure

Polyethersulfone and nylon membranes were cleaned as described inExample 2 above. The membranes were coated with the antimicrobial resinsolution described in Example 1 (1a or 1b) and dried. The coatings thenwere crosslinked by heating at 120° C. The resulting crosslinkedcoatings were rinsed with water to remove any unbound polymer, wererinsed with acidified water or buffer [pH2-2.5], followed by anotherwater rinse, then dried. Silver was deposited into the crosslinkedpolymer matrix by immersing the coated membrane in a 0.05% solution of asilver iodide/potassium iodide complex in aqueous alcohol.

Unbound silver iodide was removed by an ethanol wash. The membrane wasrinsed with water, then dried at 70° C. for 30 minutes.

Example 4 Contact Killing Ability

The coated articles described in Example 2 and the membranes describedin Example 3 were exposed to a variety of bacteria from the followinggenera: Pseudomonas, Staphylococcus, Serratia, Klebsiella, Bacillus,Enterococcus and Aspergillus, and a fungus from the genus Candida. Thespecies of microorganisms used are listed in Tables 1 and 2.

The articles and membranes were incubated with the microorganisms at35-30° C. for at least 20 hours, and for as long as 504 hours (21 days).The results are shown in Tables 1 and 2:

TABLE 1 Biocidal activity of treated surface Time to Kill OrganismChallenge Complete kill at 30° C. Pseudomonas dimunata 10⁶ CFU/mL 20hours Pseudomonas cepacia 10⁶ CFU/mL 20 hours Staphylococcus aureus 10⁶CFU/mL 20 hours Serratia marcescens 10⁶ CFU/mL 20 hours Escherichia coli10⁶ CFU/mL 20 hours Klebsiella pneumoniae 10⁶ CFU/mL 20 hours Bacillussubtilis 10⁶ CFU/mL 20 hours Bacillus cerius 10⁶ CFU/mL 20 hoursStaphylococcus epidermidis 10⁵ CFU/mL 72 hours Enterococcus faecalzs 10⁶CFU/mL 20 hours Candida albicans 10⁶ CFU/mL 168 hours  Aspergillus niger10⁵ CFU/mL no growth* *21 days at 25° C.

TABLE 2 Bacterial Grow through challenge of treated membranes with 10⁶CFU/mL Pseudomonas aeruginosa In PBS at 30° C. Membrane Type Days inTest Days to Failure Nylon Membrane, 0.2 μm, 32 30 untreated controlNylon Membrane, 0.2 μm, 54 None AMS coated Polyether sulfone 0.2 μm, 5 3 untreated control Polyether sulfone 0.2 μm, 70 None AMS coated AMS =antimicrobial surface

Example 5 Kinetics of Antimicrobial Action

The coating acts upon contact with the micro-organism, firstintercalating into the cell membrane and second transferring thebio-toxic agent directly to the contacting organism. The following timeto kill experiment was performed on polyethylene tubes with variousdiameters coated with the PHMB-BMDGA-silver coating described inExample 1. Coatings were applied as described in Example 2 for thecontact lens cases. The tubes were inoculated with predetermined volumesof initial concentrations of up to 10⁹ cfu/mL of Pseudomonas aeruginosa(ATCC#9027) in PBS and incubated at 30° C. for 20 hours. At various timepoints tubes were sampled and the micro-organism was plated and counted.The treated tubes demonstrated significant antibacterial activity evenwhen volume to surface ratios (S/V) exceeded 4:1. The results are shownin FIG. 3.

Additional evidence for the contact killing mechanism was provided bythe following experiment. Polypropylene tubes were coated as describedin Example 2 for contact lens cases. The coated tubes and untreatedcontrols were challenged with 10⁶ cfu/ml of Pseudomonas aeruginosa inPBS at 30° C. for 20 hours. An organism count by standard platingtechniques showed no viable organisms, i.e., a complete elimination (6log decrease) compared to the untreated tubes.

The solution containing the dead bacteria from the coated tubes wasdigested in 0.1M nitric acid and analyzed for the presence of silver.Silver concentration was found to be about 600 ppb. A coated tubecontaining blank PBS (no bacteria) incubated for the same time showed nodetectable silver in the solution (less than 10 ppb).

Example 6 Non-Leachability of the Coatings

To simulate an aging of approximately 1 year at ambient temperature,membranes with very large surface area were coated as described inExample 3. The coated membranes were immersed in water, isotonic salineand phosphate buffered saline solutions at 70° C. for 5 days. The testsolutions were analyzed for elutables by spectroscopic methods withsensitivities less than 10 parts per billion (ppb) of activeingredients, i.e., PHMB, BMDGA, silver and iodide. The following levelswere found:

Silver: less than 10 ppb (below detection limit) PHMB: less than 100 ppb(below detection limit) BMGDA: less than 300 ppb (below quantitationlimit) Iodide: less than 50 ppb (below quantitation limit)

These analytical results were further confirmed by testing the contactsolutions to demonstrate that they show no antimicrobial activity bychallenging them with silver sensitive Escherichia coli (ATCC # 8739) ata concentration of 10⁶ cfu/mL. No decrease in numbers of themicroorganism was detected after 20 hours.

Example 7 Toxicity

For assessing mammalian cell toxicity, polypropylene tubes coated asdescribed in Example 2 for contact lens cases, were aged in phosphatebuffered saline at 50° C. for 48 hours. Test solutions were evaluatedfor toxicity with mouse fibroblast cells and showed no toxicity to thecells.

Example 8 Mechanical Strength

Treated surfaces coated as described in Example 2 were subjected toSutherland rub test with 4PSI for 50 strokes and remained viable whilethe rubbing surface did not show antimicrobial activity.

Example 9 Inertness

The coating remains fully inert and bio-active after exposure to avariety of physical and chemical stresses:

-   -   Low temperature (−15° C.), 24 hours    -   Ethanol and boiling water, 1 hour    -   Prolonged exposure to acidic and basic solutions of varying pH        (4-10), 12 hours    -   High ionic strength solution (2% sodium chloride), 24 hours    -   Autoclaving (121° C. for 15 minutes)    -   Long term exposure to urine (35° C. for 7 days)    -   Challenged with 0.7% human serum albumin in phosphate buffered        saline in accelerated aging tests (noted a small increase in        non-bioavailable silver elutables due to protein complexation)        at 80° C. for 72 hours    -   Exposure to blood products    -   Worn by human volunteers for a 3 day period. No skin reaction        was noted

Example 10 Surface Bio-Activity

The coating kills micro-organisms on contact—but is non-toxic tomammalian cells. In laboratory testing, treated surfaces (polypropylene,polyethylene, nylon and polyethersulfone) effectively eliminated allhuman pathogens tested—including bacteria, yeast and fungi.

-   -   Bacillus cereus (ATCC# 11778)—10⁶ cfu/mL in 20 hours    -   Escherichia coli (ATCC#8739)—106 cfu/mL in 20 hours    -   Pseudomonas aeruginosa (ATCC#9027)—10⁶ cfu/mL in 20 hours    -   Pseudomonas cepacia (ATCC#25416)—10⁵ cfu/mL in 20 hours    -   Pseudomonas diminuta (ATCC#19146)—10⁶ cfu/mL in 20 hours    -   Klebsiella pneumoniae (ATCC#13883)—10⁶ cfu/mL in 20 hours    -   Staphylococus aureus (ATCC#6538)—10⁶ cfu/mL in 20 hours    -   Serratia marcescens (ATCC#8100)—10⁶ cfu/mL in 20 hours    -   Enterococcus faecalis (ATCC#19433)—10⁶ cfu/mL in 20 hours    -   Staphylococus epidermidis (ATCC# 12228)—10⁵ cfu/mL in 72 hours    -   Candida albicans (ATCC#10231)—10⁵ cfu/mL in 168 hours

Surfaces coated as described in Example 2 were challenged with thesemicroorganisms in the initial concentrations indicated. Themicroorganisms were suspended in phosphate buffered saline and wereallowed to remain in contact with the treated surfaces for extendedperiods at 30° C. The solutions were then analyzed using standardplating methods. While organism growth was documented on untreatedsurfaces, the microorganisms were completely eliminated on the Surfacinetreated samples in the specified time period. These results wereconfirmed in thousands of tests conducted over three years.

In addition, the treated surfaces were tested against Aspergillus niger.No fungal growth was detected over the 28 day test period.

Example 11 Prevention of Bio-Film Formation

To determine efficacy against bio-film formation, polyurethane tubescoated as described in Example 2 and untreated tubes were challengedwith a mixture of the following micro-organisms, incubated in a 1%synthetic growth medium at room temperature.: Pseudomonas diminuta(ATCC#19146), Pseudomonas aeruginosa (ATCC#9027), Klebsiella pneumoniae(ATCC#13883), Bacillus cereus (ATCC#11778), Escherichia Coli(ATCC#8739), Staphylococcus aureus (ATCC#6538). Within 24 hours, themicro-organisms in untreated tubes had grown from an initialconcentration of 10⁴ cfu/mL to an average of 3×10⁵ cfu/mL. The treatedtubes had no viable micro-organisms.

The tubes were then washed and refilled with water. Eight days later,the untreated tubes still yielded 10⁵ cfu/mL (resulting from thebio-film established during the first day of incubation) while thetreated tubes yielded no micro-organisms.

Example 12 Antibiotic Resistant Bacteria

Untreated and treated (as described in Example 2) surfaces werechallenged with 10⁶ cfu/ml of methicillin and neomycin resistant strainof Staphylococcus aureus (ATCC#33592). The micro-organism was suspendedin phosphate buffered saline and were allowed to remain in contact withthe surfaces. Within 20 hours, treated surfaces had no viable organisms,whereas the number of viable organisms on untreated surfaces remainedunchanged.

Equivalents

Those skilled in the art will be able to ascertain, using no more thanroutine experimentation, many equivalents of the specific embodiments ofthe invention described herein. These and all other equivalents areintended to be encompassed by the following claims.

1. An article of manufacture comprising an adherent antimicrobialcoating comprising a nitrogen-containing polycationic polymer matriximmobilized on a surface of the article of manufacture, and anantimicrobial metallic material bound to the matrix such that theantimicrobial coating does not release biocidal amounts of elutablesinto the surrounding ambient liquid for at least 5 days.
 2. The articleof claim 1 wherein the nitrogen-containing polycationic polymer matrixcomprises benzalkonium groups.
 3. The article of claim 1 wherein theantimicrobial metallic material is selected from the group consisting ofa metal, a metal salt, a metal complex, a metal alloy, and mixturesthereof.
 4. The article of claim 3 wherein the metallic materialcomprises silver.
 5. The article of claim 3 wherein the mixturecomprises silver and copper.
 6. The article of claim 3 wherein the metalsalt is silver iodide.
 7. The article of claim 1 wherein thenitrogen-containing polycationic polymer matrix is crosslinked with acrosslinking agent.
 8. The article of claim 7, wherein the crosslinkingagent is selected from the group consisting of isocyanates, carboxylicacids, acid chlorides, acid anhydrides, succinimidyl ether aldehydes,ketones, alkyl methanesulfonates, alkyl trifluoromethanesulfonates,alkyl para-toluenemethanesulfonates, alkyl halides, and epoxides.
 9. Thearticle of claim 7 wherein the crosslinking agent isN,N-methylene-bis-diglycidylaniline.
 10. The article of claim 1 whereinthe article is a medical device, a personal care product, or a consumerproduct.
 11. The article of claim 1 wherein the article is a medicaldevice selected from the group consisting of surgical gloves, surgicalinstruments, dental care instruments, dental consoles, instrument trays,catheters, urological devices, blood collection and transferringdevices, devices from for implanting in a patient, urine collectiondevices, ophthalmic devices, intraocular lenses, tracheotomy devices,topical disinfectants and wound dressings.
 12. The article of claim 10wherein the personal care product is selected from the group consistingof hair care items, toothbrushes, dental floss, dental implements,contact lenses, contact lens storage cases, baby care items, child careitems, bathroom implements, bed linens, towels and washcloths.
 13. Thearticle of claim 10, wherein the consumer product is selected from thegroup consisting of kitchen implements, trash containers, disposabletrash bags and cutting boards.
 14. The article of manufacture of claim1, wherein the antimicrobial coating is immobilized on the surface bycovalent bonding, ionic interaction, coulombic interaction, hydrogenbonding, or crosslinking.
 15. The article of manufacture of claim 1,wherein the antimicrobial coating is immobilized on the surface via afunctional group of the nitrogen-containing polycationic polymer. 16.The article of manufacture of claim 1, wherein the functional groupselected from the group consisting of a thiol group, a hydroxy group, anamine group, a halogen, an epoxy group, an alkyl group, and an alkoxygroup.
 17. An article of manufacture comprising an adherentantimicrobial coating comprising a nitrogen-containing polycationicpolymer matrix immobilized on a surface of the article of manufacture bycovalent bonding, ionic interaction, coulombic interaction, hydrogenbonding, or cross-linking, and an antimicrobial metallic material boundor attached to the matrix such that the antimicrobial coating does notrelease biocidal amounts of elutables into the surrounding ambientliquid for at least 5 days.
 18. An article of manufacture comprising anadherent antimicrobial coating comprising a nitrogen-containingpolycationic polymer matrix immobilized on a surface of the article ofmanufacture, the nitrogen-containing polycationic polymer matrix beingfunctionalized to enable immobilization on the surface, and anantimicrobial metallic material bound to the matrix such that theantimicrobial coating does not release biocidal amounts of elutablesinto the surrounding ambient liquid for at least 5 days.
 19. The articleof manufacture of claim 18, wherein the nitrogen-containing polycationicpolymer is functionalized with a thiol group, a hydroxy group, an aminegroup, a halogen, an epoxy group, an alkyl group, an alkoxy group, andmixtures thereof.